A main mechanism by which HPV 16 exerts oncogenic effects is through the expression of two viral early genes, E6 and E7. Therefore, factors that regulate the expression of E6 and E7 are likely to influence transformation of HPV 16 infections to higher grades of cervical intraepithelial neoplasia (CIN 2+) or invasive cervical cancer (ICC). The degree of HPV 16 DNA methylation (DNAm) has been shown to influence its gene expression, viral life cycle, persistence and replicative behavior. Our recent studies demonstrated the following: 1) A higher degree of HPV 16 DNAm assayed in exfoliated cells (ECs) from the cervix was associated with 79% reduced likelihood of being diagnosed with CIN 2+ 2) The degree of HPV 16 DNAm showed the best positive predictive value (PPV) for identifying women with CIN 2+, compared to women tested positive for any carcinogenic HPV type or tested positive for abnormal pap, two tests commonly used by Health Departments to identify high risk women 3) HPV 16-positive women with lower circulating concentrations of folate were nine times more likely to be diagnosed with CIN 2+ compared to HPV 16-negative women with higher folate. Because HPV uses the human methylation machinery for its DNAm, the same factors which influence DNAm in human cells, namely the availability of methyl groups generated by the folate metabolic pathway, may also have the ability to alter the degree of HPV 16 DNAm. With this background, the aims of the study are as follows: Aim 1. To evaluate the association between the degree of HPV 16 DNAm and CIN using DNA extracted from paired samples of ECs and cervical biopsies at sites identified in our published study (position 7862 of the HPV 16 E6 enhancer and positions 31, 37, 43, 52 and 58 of the HPV 16 E6 promoter) and CIN 2+, in order to replicate our findings in a separate, larger group of women. This aim will be tested among samples already collected from 300 women diagnosed with CIN 2+ (cases) and 300 women diagnosed with d CIN 1 (non-cases) Aim 2. To evaluate the association between the degree of HPV 16 DNAm at the same sites as in aim 1 and circulating concentrations of folate, in order to assess the influence of exposure to different levels of folate as a result of the US folic acid fortification program. This aim will be tested among samples already collected from 300 women diagnosed with d CIN 1. The study will provide a unique opportunity to determine the diagnostic value of testing the degree of HPV 16 DNAm for identifying women at high risk for developing HPV 16 associated CIN 2+. Further, the study integrates an important population-wide environmental exposure (folate) and molecular data by testing the hypothesis that exposure to folate may have the ability to alter the degree of HPV 16 DNAm and modify the HPV 16 associated risk of developing CIN in the US post-folic acid fortification era. PUBLIC HEALTH RELEVANCE: The purpose of the study is to assess whether specific methylation changes in HPV 16 can be used as a biomarker for enhanced risk of advanced cervical intraepithelial neoplasia and to determine whether folic acid levels predict the degree of HPV 16 methylation.